It is known that HDL receives cholesterol from various tissues, including the walls of blood vessels with arterial sclerosis, and it is thus associated with removal of cholesterol accumulated in cells. Also, HDL is a preventive factor for onset of various arteriosclerotic diseases, including coronary artery sclerosis, and the HDL level in the blood serves as a useful indicator for onset of an arteriosclerotic disease.
HDL is a composite of lipid components, such as a protein referred to as an “apoprotein,” phospholipid, cholesterol, and neutral fat. One such lipid component is apolipoprotein E (apoE), and HDL can be classified as an apoE-containing HDL subfraction or an apoE-deficient HDL subfraction based on apoE content. ApoE-containing HDL has strong cholesterol efflux capacity and anti-platelet effects, and, among various types of HDLs, it has drawn attention as a very good lipoprotein. In recent years, a CETP inhibitor that elevates HDL-C has been expected as a lipid-lowering agent following statin. CETP inhibitors are known to mainly elevate the apoE-containing HDL among various types of HDLs.
As methods for assaying cholesterol in HDL, for example, a method comprising separating HDL from other lipoproteins via ultracentrifugation and assaying cholesterol and a method comprising separating HDL via electrophoresis and staining lipids to assay the intensity of color development have been known. These methods, however, are disadvantageous due to complicated procedures, the incapability of simultaneous processing of many test samples, and other problems. Thus, such methods are hardly ever employed on a routine basis.
An example of a method for assaying cholesterol in HDL is a method comprising adding a precipitating agent to a test sample to coagulate lipoproteins other than HDL, removing the resulting coagulate via centrifugation, and assaying cholesterol in the supernatant selectively containing the separated HDL. According to such method, reactivity to an HDL subfraction is known to vary depending on the type of precipitating agent used. According to a method involving the use of phosphotungstic acid-magnesium, dextran sulfate-magnesium, or heparin-calcium as a precipitating agent, apoE-containing HDL coagulates with lipoproteins, such as VLDL and LDL, and it is removed as a precipitated fraction via centrifugation. Thus, apoE-containing HDL cannot be assayed as an HDL fraction. According to a method involving the use of heparin-manganese or polyethylene glycol (PEG), apoE-containing HDL does not coagulate, and it is assayed as HDL. While a method involving the use of a precipitating agent to separately quantify HDL can be carried out more simply than ultracentrifugation or electrophoresis, this method comprises addition of a precipitating agent and separation. Thus, it is insufficient in terms of convenience and the necessity for relatively large quantities of test samples.
In recent years, a method of quantification of HDL-C with the use of an automatic analyzer without pretreatment with a precipitating agent has been known as a simple method of HDL-C quantification. For example, a method in which cholesterol esterase or cholesterol oxidase is chemically modified and cholesterol in HDL is captured in a specific manner in the presence of an inclusion compound, such as cyclodextrin (see Patent Document 1), a method in which a coagulate or complex with lipoproteins other than HDL is formed and cholesterol in HDL is then captured via an enzymatic reaction (see Patent Documents 2 and 3), and a method involving the use of a surfactant with an HLB of 13 to 14, which specifically acts on HDL (see Patent Document 4), have been known.